RESUMEN
HIV Tat is an essential protein required for the transcription elongation of HIV genome. It has been shown that Tat can be degraded by either proteasome or autophagy pathways. In this study, it was shown that proteasome inhibitor MG132 could significantly prevent HIV Tat protein degradation in Tat over-expressing HeLa cells but it had a moderate effect in preventing Tat protein degradation in Jurkat T cells. A screening of the available UBE2 siRNA family identified that UBE2R1 had a high repressive effect on Tat protein but not on Tat mRNA level. This study further showed that RNF20 might not be the E3 ligase of Tat but was required to maintain a high level of H2B-monoubiquitylation (H2Bub1) on HIV-1 genome for efficient elongation. Overall, our study indicated that UBE2R1 might be the potential ubiquitin E2 ligase for HIV Tat protein turnover and RNF20 regulated HIV expression in the transcription elongation level.
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Co-transcriptional histone modifications are a ubiquitous feature of RNA polymerase II (RNAPII) transcription, with profound but incompletely understood effects on gene expression. Unlike the covalent marks found at promoters, which are thought to be instructive for transcriptional activation, these modifications occur in gene bodies as a result of transcription, which has made elucidation of their functions challenging. Here we review recent insights into the regulation and roles of two such modifications: monoubiquitylation of histone H2B at lysine 120 (H2Bub1) and methylation of histone H3 at lysine 36 (H3K36me). Both H2Bub1 and H3K36me are enriched in the coding regions of transcribed genes, with highly overlapping distributions, but they were thought to work largely independently. We highlight our recent demonstration that, as was previously shown for H3K36me, H2Bub1 signals to the histone deacetylase (HDAC) complex Rpd3S/Clr6-CII, and that Rpd3S/Clr6-CII and H2Bub1 function in the same pathway to repress aberrant antisense transcription initiating within gene coding regions. Moreover, both of these histone modification pathways are influenced by protein phosphorylation catalyzed by the cyclin-dependent kinases (CDKs) that regulate RNAPII elongation, chiefly Cdk9. Therefore, H2Bub1 and H3K36me are more tightly linked than previously thought, sharing both upstream regulatory inputs and downstream effectors. Moreover, these newfound connections suggest extensive, bidirectional signaling between RNAPII elongation complexes and chromatin-modifying enzymes, which helps to determine transcriptional outputs and should be a focus for future investigation.
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Código de Histonas , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , UbiquitinaciónRESUMEN
As a member of the 11-gene "death-from-cancer" gene expression signature, ubiquitin-specific protease 22 (USP22) has been considered an oncogene in various human malignancies, including colorectal cancer (CRC). We recently identified an unexpected tumor-suppressive function of USP22 in CRC and detected intestinal inflammation after Usp22 deletion in mice. We aimed to investigate the function of USP22 in intestinal inflammation as well as inflammation-associated CRC. We evaluated the effects of a conditional, intestine-specific knockout of Usp22 during dextran sodium sulfate (DSS)-induced colitis and in a model for inflammation-associated CRC. Mice were analyzed phenotypically and histologically. Differentially regulated genes were identified in USP22-deficient human CRC cells and the occupancy of active histone markers was determined using chromatin immunoprecipitation. The knockout of Usp22 increased inflammation-associated symptoms after DSS treatment locally and systemically. In addition, Usp22 deletion resulted in increased inflammation-associated colorectal tumor growth. Mechanistically, USP22 depletion in human CRC cells induced a profound upregulation of secreted protein acidic and rich in cysteine (SPARC) by affecting H3K27ac and H2Bub1 occupancy on the SPARC gene. The induction of SPARC was confirmed in vivo in our intestinal Usp22-deficient mice. Together, our findings uncover that USP22 controls SPARC expression and inflammation intensity in colitis and CRC.
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Chromosome instability (CIN) is an enabling feature of oncogenesis associated with poor patient outcomes, whose genetic determinants remain largely unknown. As mitotic chromatin compaction defects can compromise the accuracy of chromosome segregation into daughter cells and drive CIN, characterizing the molecular mechanisms ensuring accurate chromatin compaction may identify novel CIN genes. In vitro, histone H2B monoubiquitination at lysine 120 (H2Bub1) impairs chromatin compaction, while in vivo H2Bub1 is rapidly depleted from chromatin upon entry into mitosis, suggesting that H2Bub1 removal may be a pre-requisite for mitotic fidelity. The deubiquitinating enzyme USP22 catalyzes H2Bub1 removal in interphase and may also be required for H2Bub1 removal in early mitosis to maintain chromosome stability. In this study, we demonstrate that siRNA-mediated USP22 depletion increases H2Bub1 levels in early mitosis and induces CIN phenotypes associated with mitotic chromatin compaction defects revealed by super-resolution microscopy. Moreover, USP22-knockout models exhibit continuously changing chromosome complements over time. These data identify mitotic removal of H2Bub1 as a critical determinant of chromatin compaction and faithful chromosome segregation. We further demonstrate that USP22 is a CIN gene, indicating that USP22 deletions, which are frequent in many tumor types, may drive genetic heterogeneity and contribute to cancer pathogenesis.
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Numerous epigenetic alterations are observed in cancer cells, and dysregulation of mono-ubiquitination of histone H2B (H2Bub1) has often been linked to tumorigenesis. H2Bub1 is a dynamic post-translational histone modification associated with transcriptional elongation and DNA damage response. Histone H2B monoubiquitination occurs in the site of lysine 120, written predominantly by E3 ubiquitin ligases RNF20/RNF40 and deubiquitinated by ubiquitin specific peptidase 22 (USP22). RNF20/40 is often altered in the primary tumors including colorectal cancer, breast cancer, ovarian cancer, prostate cancer, and lung cancer, and the loss of H2Bub1 is usually associated with poor prognosis in tumor patients. The purpose of this review is to summarize the current knowledge of H2Bub1 in transcription, DNA damage response and primary tumors. This review also provides novel options for exploiting the potential therapeutic target H2Bub1 in personalized cancer therapy.
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Regulación Neoplásica de la Expresión Génica , Histonas/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Ubiquitinadas/fisiología , Carcinoma/etiología , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/terapia , Daño del ADN , Reparación del ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Progresión de la Enfermedad , Humanos , Proteínas de Neoplasias/genética , Neoplasias/etiología , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisión , Elongación de la Transcripción Genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Modification of the cancer-associated chromatin landscape in response to therapeutic DNA damage influences gene expression and contributes to cell fate. The central histone mark H2Bub1 results from addition of a single ubiquitin on lysine 120 of histone H2B and is an important regulator of gene expression. Following treatment with a platinum-based chemotherapeutic, there is a reduction in global levels of H2Bub1 accompanied by an increase in levels of the tumor suppressor p53. Although total H2Bub1 decreases following DNA damage, H2Bub1 is enriched downstream of transcription start sites of specific genes. Gene-specific H2Bub1 enrichment was observed at a defined group of genes that clustered into cancer-related pathways and correlated with increased gene expression. H2Bub1-enriched genes encompassed fifteen p53 target genes including PPM1D, BTG2, PLK2, MDM2, CDKN1A and BBC3, genes related to ERK/MAPK signalling, those participating in nucleotide excision repair including XPC, and genes involved in the immune response and platinum drug resistance including POLH. Enrichment of H2Bub1 at key cancer-related genes may function to regulate gene expression and influence the cellular response to therapeutic DNA damage.
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Cromatina/metabolismo , Daño del ADN/genética , Transducción de Señal/genética , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Cisplatino/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Mono-ubiquitination on H2B (H2Bub1) is an evolutionarily conserved histone post-translational modification implicated in various important physiological processes including DNA replication, transcription activation, and DNA damage repair. The Bre1/Rad6 ubiquitination machinery is currently considered to be the sole writer of H2Bub1, but the mechanistic basis by which it operates is unclear. Recently, the RING-type E3 ligase Bre1 was proposed to associate with the E2 enzyme Rad6 through a novel interaction between Bre1 RBD (Rad6 binding domain) and Rad6; and the RING domain of Bre1 that is responsible for the nucleosomal acidic patch binding also interacts with Rad6 to stimulate its catalytic activity. Recent discoveries have yielded evidence for the phenomenon of liquid-liquid phase separation in the context of H2Bub1, and its regulation by other histone post-translational modifications. This review summarizes current knowledge about Bre1/Rad6-mediated H2B ubiquitination, including the physiological functions and the molecular basis for writing and regulation of this central histone ubiquitin mark. Possible models for the Bre1/Rad6 machinery bound to nucleosomes bearing different modifications in the writing step are also disclosed.
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Histonas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Humanos , Metilación , Nucleosomas/metabolismo , Relación Estructura-Actividad , Elongación de la Transcripción Genética , Activación Transcripcional , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Ovarian cancer is the most lethal gynecologic malignancy in the United States. The mortality of this disease is primarily attributed to challenges in early detection and therapeutic resistance. Recent studies indicate that the majority of high-grade serous ovarian carcinomas (HGSCs) originate from aberrant fallopian tube epithelial (FTE) cells. This shift in thinking about ovarian cancer pathogenesis has been met with an effort to identify the early genetic and epigenetic changes that underlie the transformation of normal FTE cells and prompt them to migrate and colonize the ovary, ultimately giving rise to aggressive HGSC. While identification of these early changes is important for biomarker discovery, the emergence of epigenetic alterations in FTE chromatin may also provide new opportunities for early detection, prevention, and therapeutic intervention. Here we provide a comprehensive overview of the current knowledge regarding early epigenetic reprogramming that precedes HGSC tumor development, the way that these alterations affect both intrinsic and extrinsic tumor properties, and how the epigenome may be targeted to thwart HGSC tumorigenesis.
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Biomarcadores de Tumor/genética , Carcinoma Epitelial de Ovario/genética , Epigénesis Genética , Epigenoma , Células Epiteliales/patología , Trompas Uterinas/patología , Neoplasias Quísticas, Mucinosas y Serosas/genética , Neoplasias Ováricas/genética , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/terapia , Células Epiteliales/metabolismo , Trompas Uterinas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Quísticas, Mucinosas y Serosas/metabolismo , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neoplasias Quísticas, Mucinosas y Serosas/terapia , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Fenotipo , Microambiente TumoralRESUMEN
Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+ -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.
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Factores de Transcripción GATA/genética , Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , ARN sin Sentido/genética , Proteínas de Schizosaccharomyces pombe/genética , Ubiquitinación , Segregación Cromosómica , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Factores de Transcripción GATA/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro. METHODS: We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK. RESULTS: Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis. CONCLUSIONS: Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.
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Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica/métodos , Silenciador del Gen , Ubiquitina-Proteína Ligasas/genética , Apoptosis , Sistemas CRISPR-Cas , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HT29 , Histonas/metabolismo , Humanos , UbiquitinaciónRESUMEN
Diabetes is characterized by a loss of ß-cell mass, and a greater understanding of the transcriptional mechanisms governing ß-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA-binding protein 3 (SSBP3) regulates the genes necessary for ß-cell function, but few proteins are known to interact with this complex in ß-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse ß-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for ß-cell function. We observed that Rnf20 and Rnf40 depletion reduces ß-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate ß-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.
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Proteínas con Dominio LIM/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Histonas/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Proteínas con Dominio LIM/química , Proteínas con Homeodominio LIM/antagonistas & inhibidores , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones , Unión Proteica , Dominios Proteicos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
BACKGROUND AND AIMS: Inflammatory bowel diseases are linked to an increased risk of developing colorectal cancer [CRC]. Previous studies suggested that the H2B ubiquitin ligase RING finger protein-20 [RNF20] inhibited inflammatory signaling mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB]. However, the role of RNF40, the obligate heterodimeric partner of RNF20, in the context of inflammation and CRC has not been addressed. Here, we examined the effect of RNF40 loss on CRC cells in vitro and on inflammation and inflammatory signaling in vitro and in vivo. METHODS: We evaluated H2Bub1 levels in human and murine colorectal tumors by immunohistochemistry. Moreover, we correlated H2Bub1 and RNF40 levels in vivo and assessed the consequences of RNF40 depletion on cellular phenotype and gene expression in CRC cells in vitro. Finally, we examined the effect of a colon-specific loss of Rnf40 in a murine model of colitis, and assessed both local and systemic inflammation-associated consequences. RESULTS: In vitro studies revealed that the tumorigenic phenotype of CRC cells decreased after RNF40 depletion and displayed gene expression changes related to chromosome segregation and DNA replication, as well as decreased induction of several NF-κB-associated cytokines. This effect was associated with decreased nuclear localization of NF-κB following tumor necrosis factor alpha treatment. Consistently, the colon-specific loss of Rnf40 exerted a protective local, as well as systemic, effect following acute colitis. CONCLUSIONS: Our findings suggest that RNF40 plays a central role in the maintenance of tumorigenic features and inflammatory signaling by promoting nuclear NF-κB activity.
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Colitis/genética , Colitis/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Carcinogénesis/genética , Núcleo Celular/metabolismo , Proliferación Celular/genética , Segregación Cromosómica , Replicación del ADN , Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Ratones , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Histone H2B lysine 123 mono-ubiquitination (H2Bub1), catalyzed by Rad6 and Bre1 in Saccharomyces cerevisiae, modulates chromatin structure and affects diverse cellular functions. H2Bub1 plays roles in telomeric silencing and telomere replication. Here, we have explored a novel role of H2Bub1 in telomere protection at uncapped telomeres in yku70Δ and cdc13-1 cells. Deletion of RAD6 or BRE1, or mutation of H2BK123R enhances the temperature sensitivity of both yku70Δ and cdc13-1 telomere capping mutants. Consistently, BRE1 deletion increases accumulation of telomeric single-stranded DNA (ssDNA) in yku70Δ and cdc13-1 cells, and EXO1 deletion improves the growth of yku70Δ bre1Δ and cdc13-1 bre1Δ cells and decreases ssDNA accumulation. Additionally, deletion of BRE1 exacerbates the rate of entry into senescence of yku70Δ mre11Δ cells with telomere defects, and increases the recombination of subtelomeric Y' element that is required for telomere maintenance and survivor generation. Furthermore, Exo1 contributes to the abrupt senescence of yku70Δ mre11Δ bre1Δ cells, and Rad51 is essential for Y' recombination to generate survivors. Finally, deletion of BRE1 or mutation of H2BK123R results in nucleosome instability at subtelomeric regions. Collectively, this study provides a mechanistic link between H2Bub1-mediated chromatin structure and telomere protection after telomere uncapping.
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Exodesoxirribonucleasas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telómero/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , ADN de Cadena Simple/metabolismo , Nucleosomas/metabolismo , Recombinación GenéticaRESUMEN
Histone H2B monoubiquitination plays a critical role in the regulation of gene transcription. Deregulation of H2B monoubiquitination contributes to human pathologies, such as cancer. Here we report that human USP36 is a novel H2Bub1 deubiquitinase. We show that USP36 interacts with H2B and deubiquitinates H2Bub1 in cells and in vitro. Overexpression of USP36 markedly reduced the levels of H2Bub1 in cells. Using the p21 gene as a model, we demonstrate that depletion of USP36 increases H2Bub1 at the p21 locus, primarily within its gene body. Consistently, knockdown of USP36 induced the expression of p21 and inhibits cell proliferation. Together, our results reveal USP36 as a novel H2B deubiquitinase and shed light on its additional functions in regulating gene expression.
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Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación/fisiología , Secuencia Conservada , Enzimas Desubicuitinizantes/genética , Endopeptidasas/genética , Activación Enzimática , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Especificidad por Sustrato , Ubiquitina Tiolesterasa/genéticaRESUMEN
Ubiquitin-Specific Peptidase 22 (USP22) is a ubiquitin hydrolase, notably catalyzing the removal of the mono-ubiquitin moiety from histone H2B (H2Bub1). Frequent overexpression of USP22 has been observed in various cancer types and is associated with poor patient prognosis. Multiple mechanisms have been identified to explain how USP22 overexpression contributes to cancer progression, and thus, USP22 has been proposed as a novel drug target in cancer. However, gene re-sequencing data from numerous cancer types show that USP22 expression is frequently diminished, suggesting it may also harbor tumor suppressor-like properties. This review will examine the current state of knowledge on USP22 expression in cancers, describe its impact on H2Bub1 abundance and present the mechanisms through which altered USP22 expression may contribute to oncogenesis, including an emerging role for USP22 in the maintenance of genome stability in cancer. Clarifying the impact aberrant USP22 expression and abnormal H2Bub1 levels have in oncogenesis is critical before precision medicine therapies can be developed that either directly target USP22 overexpression or exploit the loss of USP22 expression in cancer cells.
RESUMEN
Histone H2B monoubiquitination (H2Bub1) is recognized as a regulatory mechanism that controls a range of cellular processes. We previously showed that H2Bub1 was involved in responses to biotic stress in Arabidopsis. However, the molecular regulatory mechanisms of H2Bub1 in controlling responses to abiotic stress remain limited. Here, we report that HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 played important regulatory roles in response to salt stress. Phenotypic analysis revealed that H2Bub1 mutants confer decreased tolerance to salt stress. Further analysis showed that H2Bub1 regulated the depolymerization of microtubules (MTs), the expression of PROTEIN TYROSINE PHOSPHATASE1 (PTP1) and MAP KINASE PHOSPHATASE (MKP) genes - DsPTP1, MKP1, IBR5, PHS1, and was required for the activation of mitogen-activated protein kinase3 (MAP kinase3, MPK3) and MPK6 in response to salt stress. Moreover, both tyrosine phosphorylation and the activation of MPK3 and MPK6 affected MT stability in salt stress response. Thus, the results indicate that H2Bub1 regulates salt stress-induced MT depolymerization, and the PTP-MPK3/6 signalling module is responsible for integrating signalling pathways that regulate MT stability, which is critical for plant salt stress tolerance.
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Arabidopsis/metabolismo , Histonas/metabolismo , Microtúbulos/metabolismo , Polimerizacion , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Ubiquitinación , Adaptación Fisiológica , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Mutación/genética , Fenotipo , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica/efectos de los fármacos , Estrés Fisiológico/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: Monoubiquitination of H2B (H2Bub1) is a largely enigmatic histone modification that has been linked to transcriptional elongation. Because of this association, it has been commonly assumed that H2Bub1 is an exclusively positively acting histone modification and that increased H2Bub1 occupancy correlates with increased gene expression. In contrast, depletion of the H2B ubiquitin ligases RNF20 or RNF40 alters the expression of only a subset of genes. RESULTS: Using conditional Rnf40 knockout mouse embryo fibroblasts, we show that genes occupied by low to moderate amounts of H2Bub1 are selectively regulated in response to Rnf40 deletion, whereas genes marked by high levels of H2Bub1 are mostly unaffected by Rnf40 loss. Furthermore, we find that decreased expression of RNF40-dependent genes is highly associated with widespread narrowing of H3K4me3 peaks. H2Bub1 promotes the broadening of H3K4me3 to increase transcriptional elongation, which together lead to increased tissue-specific gene transcription. Notably, genes upregulated following Rnf40 deletion, including Foxl2, are enriched for H3K27me3, which is decreased following Rnf40 deletion due to decreased expression of the Ezh2 gene. As a consequence, increased expression of some RNF40-"suppressed" genes is associated with enhancer activation via FOXL2. CONCLUSION: Together these findings reveal the complexity and context-dependency whereby one histone modification can have divergent effects on gene transcription. Furthermore, we show that these effects are dependent upon the activity of other epigenetic regulatory proteins and histone modifications.
Asunto(s)
Epigénesis Genética , Regulación de la Expresión Génica , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Quinasa 9 Dependiente de la Ciclina/metabolismo , Elementos de Facilitación Genéticos , Proteína Potenciadora del Homólogo Zeste 2/genética , Fibroblastos/metabolismo , Genes Homeobox , Histonas/metabolismo , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Unión Proteica , Elongación de la Transcripción Genética , Transcripción Genética , Activación Transcripcional , UbiquitinaciónRESUMEN
It is known that signaling from the germline stem cell niche is required to maintain germline stem cell identity in Drosophila. However, it is not clear whether the germline stem-cell daughters differentiate by default (because they are physically distant from the niche) or whether additional signaling is necessary to initiate the differentiation program. Previously, we showed that ecdysteroid signaling cell non-autonomously regulates early germline differentiation via its soma-specific co-activator and co-repressor, Taiman and Abrupt. Now, we demonstrate that this regulation is modulated by the miRNA let-7, which acts in a positive feedback loop to confer ecdysone signaling robustness via targeting its repressor, the transcription factor Abrupt. This feedback loop adjusts ecdysteroid signaling in response to some stressful alterations in the external and internal conditions, which include temperature stress and aging, but not nutritional deprivation. Upon let-7 deficit, escort cells fail to properly differentiate: their shape, division, and cell adhesive characteristics are perturbed. These cells have confused cellular identity and form columnar-like rather than squamous epithelium and fail to send protrusions in between differentiating germline cysts, affecting soma-germline communication. Particularly, levels of the homophilic cell adhesion protein Cadherin, which recruits Wg signaling transducer ß-catenin, are increased in mutant escort cells and, correspondingly, in the adjacent germline cells. Readjustment of heterotypic (soma-germline) cell adhesion modulates Wg signaling intensity in the germline, which in turn regulates histone modifications that promote expression of the genes necessary to trigger early germline differentiation. Thus, our data first show the intrinsic role for Wg signaling in the germline and support a model where the soma influences the tempo of germline differentiation in response to external conditions.
RESUMEN
The loss of genome stability is an early event that drives the development and progression of virtually all tumor types. Recent studies have revealed that certain histone post-translational modifications exhibit dynamic and global increases in abundance that coincide with mitosis and exhibit essential roles in maintaining genomic stability. Histone H2B ubiquitination at lysine 120 (H2Bub1) is regulated by RNF20, an E3 ubiquitin ligase that is altered in many tumor types. Through an evolutionarily conserved trans-histone pathway, H2Bub1 is an essential prerequisite for subsequent downstream dimethylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Although the role that RNF20 plays in tumorigenesis has garnered much attention, the downstream components of the trans-histone pathway, H3K4me2 and H3K79me2, and their potential contributions to genome stability remain largely overlooked. In this study, we employ single-cell imaging and biochemical approaches to investigate the spatial and temporal patterning of RNF20, H2Bub1, H3K4me2, and H3K79me2 throughout the cell cycle, with a particular focus on mitosis. We show that H2Bub1, H3K4me2, and H3K79me2 exhibit distinct temporal progression patterns throughout the cell cycle. Most notably, we demonstrate that H3K79me2 is a highly dynamic histone post-translational modification that reaches maximal abundance during mitosis in an H2Bub1-independent manner. Using RNAi and chemical genetic approaches, we identify DOT1L as a histone methyltransferase required for the mitotic-associated increases in H3K79me2. We also demonstrate that the loss of mitotic H3K79me2 levels correlates with increases in chromosome numbers and increases in mitotic defects. Collectively, these data suggest that H3K79me2 dynamics during mitosis are normally required to maintain genome stability and further implicate the loss of H3K79me2 during mitosis as a pathogenic event that contributes to the development and progression of tumors.